Construction Of Functional Nanoprobes Based On DNA Molecular Coding And Its Biosensing Application

Studies have shown that malignant diseases are closely related to the biological activity of molecules,and the expression of pathogenic genes is often regulated at the epigenetic or transcriptional level.The sensitive detection of bioactive molecules(microRNA and DNA repair enzyme activity)associated with malignant diseases in clinical fluid samples(urine,plasma or cell lysate)is of great significance for the study of disease pathogenesis,prevention and drug efficacy monitoring.However,the abundance of these substances in clinical liquid samples is very low,and the detection sensitivity of classical probes is not enough.How to develop functional probes with high sensitivity and specificity is a bottleneck problem for the detection of bioactive molecules.DNA nano-coding technology and the physical and chemical properties of DNA provide new ideas for the design of new functional probes.Among them,the multifunctional nucleic acid probe based on DNA molecular coding technology has the advantages of accurate base pairing,excellent addressibility and carrying multiple functional units.The DNA molecular coding probes is conducive to flexible design of signal transduction and signal output mode,and shows great potential in improving the affinity and signal output strength of biological systems.The traditional DNA molecule-encoded nucleic acid probe mainly relies on the base pairing characteristics for detecting target,and the fault tolerance rate of single recognition pattern is low.Moreover,the simple structure of the probe can easily be degraded in complex biological substrates,resulting in signal leakage and high background,which limits its application in life analysis.Therefore,it is urgent to develop novel DNA molecule-encoded nucleic acid nanoprobes with strong specificity,high sensitivity and stable structure to achieve high signal output in complex biological substrates.In view of the above limitations and challenges,we have developed a series of novel DNA molecule-encoded nucleic acid functional probes for sensitive detection of microRNA(miRNA)and DNA repair enzyme activity(T4 polynucleotide kinase,T4 PNK).It mainly includes the following two parts:(1)The "luminescent G-quadruplex nanostring" based on a double-strand specific nuclease-mediated tandem amplification circuit was develped for label-free specific detection of microRNA.Taking miRNA as a model,a long DNA generated from upstream RCA was designed with the antisense sequences for miRNA and was used as the coding motif.Upon recognizing miRNA,the resulting DNA–RNA permitted DSN digestion and triggered downstream two-way RCA,and generation of abundant “Gquadruplex nanostring” binding with zinc porphyrin(Zn PPIX)for label-free fluorescent responses.Compared with the currently reported methodsbased on double-stranded DNA(dsDNA)or hairpin DNA molecular probes,this strategy has unique characteristics:(ⅰ)Abundant "G-quadruplex nanostring signal output elements" and multiple the coordinated amplification coupling of Zn PPIX can ensure the sensitivity of miRNA detection;(ⅱ)The base pairing characteristics of DNA/RNA and the strong preference of DSN for perfectly matched DNA/RNA ensures its excellent selectivity;(ⅲ)The structure of "luminescent G-tetrastrand nanocirmers" is stable,and the miR-21 levels in cell extracts have been evaluated,revealing the utility of this tool for biomedical research and clinical diagnosis.(2)A “Raman signal tow”constructed by-encoded gold nanorods/sphere was used for sensitive detection of T4 PNK.Taking T4 PNK as a model,the long DNA chain obtained by the RCA reaction on the surface of the gold nanorod served as a coding motif and a chemical hook for nucleic acid functional monomer.The short DNA chain labeled with sulfhydryl and the fluorescent dye Cy3 was riveted on the long RCA-DNA chain to form multiple double-stranded fragments.Next,with the addition of gold nanospheres,stable and firm Au-S chemical bond can be formed.In this manner,and a large number of gold nanospheres and Raman tag Cy3 were closely arranged along the double-stranded RCA fragments to form a "wireless signal tower" nanoprobe.In our work,the gold nanorodsact as the base of the "signal tower",and the long RCA chain carrying Cy3 acts as the "antenna".Owing to the abundant SH sites of the RCA long chain,a large number of gold nanospheres were tied to the "antenna" and thus a high-density Raman hot spot was formed locally.This strategy provides a new strategy for using RCA long chains to encode multiple nanoparticles,which is expected to be applied to the detection system of other biologically active molecules and provide technical support for clinical early diagnosis.